Polyoxazoline Polymers
Polyoxazoline (POX) polymers have been used in cosmetic and food packaging applications. Due to good water solubility, POX also has been considered a candidate to replace polyethyleneglycol (PEG) for different biomedical-related applications (Adams, Advanced Drug Delivery Reviews 59 (2007) 1504-1520 and Mero, Journal of Controlled Release 125 (2008) 87-95).
POX polymers comprising, for example, poly (unsubstituted oxazoline) or poly (substituted oxazoline), can be produced by cationic ring opening polymerization. Commonly used water-soluble polymers are PMOX and PEOX. Under living polymerization conditions (e.g., conditions including fast initiation, slow propagation and the lack of chain termination and transfer reactions), a well defined, linear PMOX and PEOX can be produced (Tomalia et al., Macromol. 1991, 24, 1435. Kobayashi, J. Polym. Sci. Part A: Polym. Chem.: Vol. 40 (2002)); while under other synthesis conditions, branched or randomly branched PMOX and PEOX polymers can be generated (Litt, Macromol. Sci. Chem. A9(5), 703-727 (1975) and Yin, U.S. Pat. No. 7,754,500).
Under both types of reaction conditions, an electrophilic (e.g., cationic) chain end can be generated and further reacted with a nucleophilic group or a molecule containing a nucleophilic group so that the polymerization reaction can be terminated. Most known methods for terminating such a reactive chain end use a monofunctional nucleophilic group, such as those consisting of a single imino (—NH—) group, for example, a morpholine or a protected piperazine (Tomalia, U.S. Pat. No. 5,773,527 and Zhang et. al., Macromol., 2009, 42 (6) 2215-2221). That is true for the termination of linear POX polymers, such as a living linear PMOX or PEOX, where a defined chain end can be generated.
The termination of a reactive POX polymer with a polyfunctional polymer to generate star, comb, Starburst or Combburst polymers is described, for example, in Tomalia, U.S. Pat. No. 5,773,527.
However, use of a multifunctional small molecule without any protecting groups to terminate a reactive POX to generate a functional polymer with only one polymer per terminating molecule is not a preferable or a desired way to make a functionalized POX. That approach tends to produce more dimeric and multimeric POX blocks, such as, star-branched and comb-branched polymers.
Symmetrically Branched (SB) Polymers (SBP) and Asymmetrically Branched (AB) Polymers (ABP)
In recent years, dendritic polymers, including Starburst dendrimers (or Dense Star polymers) and Combburst dendrigrafts (or hyper comb-branched polymers), have been developed for a variety of applications (“Dendritic Molecules” ed. by Newkome et al., VCH, Weinheim, 1996 and “Dendrimers and Other Dendritic Polymers” ed. by Frechet & Tomalia, John Wiley & Sons, Ltd., 2001). Those polymers exhibit: (a) a well-defined core molecule, (b) at least two concentric dendritic layers (generations) with symmetrical (equal) branch junctures and (c) exterior surface groups, as described in U.S. Pat. Nos. 4,435,548; 4,507,466; 4,568,737; 4,587,329; 5,338,532; 5,527,524; and 5,714,166, and the references cited therein.
SB dendrimers also are distinctively different from the previously prepared AB dendrimers (U.S. Pat. Nos. 4,289,872; 4,360,646; and 4,410,688 of Denkewalter) which possess asymmetrical (unequal) branch junctures.
Both types of dendrimers can be produced by repetitive protecting and deprotecting procedures through either a divergent or a convergent synthetic approach. Since SB and AB dendrimers utilize small molecules as building blocks for the core and the branches, the molecular weights of such dendrimers often are precisely defined. In the case of lower generation molecules, a single molecular weight dendrimer often is obtained.
Similar to dendrimers, Combburst dendrigrafts also are constructed with a core and concentric layers with symmetrical branches through a stepwise synthetic method. In contrast to dendrimers, Combburst dendrigrafts or polymers are generated with monodisperse linear polymeric building blocks (Tomalia, U.S. Pat. No. 5,773,527 and Yin, U.S. Pat. Nos. 5,631,329 and 5,919,442). Moreover, the branch pattern also is different from that of dendrimers. For example, Combburst dendrigrafts form branch junctures along the polymeric backbones (chain branches) while Starburst dendrimers often branch at the termini (terminal branches). Due to the utilization of living polymerization techniques, the molecular weight distribution (Mw/Mn) of such polymeric building blocks (core and branches) often is narrow. As a result, Combburst dendrigrafts, produced through a graft-on-graft process, are rather well defined with an Mw/Mn often less than 1.2.
Although possessing well controlled molecular architecture, such as, well defined size, shape and surface functional groups, both dendrimers and dendrigrafts can be produced only through a large number of reiteration steps, making such useful only for academic pursuits rather than large scale commercial applications.
Dendrimers and dendrigrafts can serve as carriers for bioactive molecules, as described in U.S. Pat. Nos. 5,338,532; 5,527,524; and 5,714,166 of Tomalia for dense star polymers and U.S. Pat. No. 5,919,442 of Yin for hyper comb-branched polymers. The surface functional groups and interior void spaces of those molecules have been suggested as a basis for the carrier property, for example, due to the well-controlled, symmetrical dendritic architecture with predictable branching patterns (either symmetrical termini or polymeric chain branching) and molecular weight.
The preparation of regular (reg) asymmetrically branched polymer (reg-ABP) made of polylysine has been described, as illustrated in U.S. Pat. Nos. 4,289,872; 4,360,646; and 4,410,688.
The synthesis and mechanisms of random (ran) asymmetrically branched polymers (ran-ABP), such as, made of polyethyleneimine (PEI), have been described (see Jones et al., J. Org. Chem. 9, 125 (1944), Jones et al., J. Org. Chem. 30, 1994 (1965) and Dick et al., J. Macromol. Sci. Chem., A4 (6), 1301-1314, (1970)).
The synthesis and characterization of ran-ABP, such as made of POX, for example, PMOX or PEOX, have been described by Litt (J. Macromol. Sci. Chem. A9(5), pp. 703-727 (1975)) and Warakomski (J. Polym. Sci. Polym. Chem. 28, 3551 (1990)).
Randomly branched PEOX has been utilized to physically encapsulate protein molecules (U.S. Pat. No. 6,716,450). However, such an approach was not designed for the direct, covalent linking of ABP with bioactive materials for bioassays and drug delivery applications.
Polymer-Bioactive Material (BM) Compositions
Polymer-bioactive material (BM) compositions, such as, PEG or polyethyleneoxide (PEO)—drug compositions, including directly or indirectly linked conjugates, or physical mixtures of PEG/PEO and drug are known. Although less extensively studied, POX—drug compositions also have been reported, including a linear polymer drug composition, such as those described in Mero et al. in J. Contr. Rel. 125 (2008)87-95 and Viegas et al., Bioconj. Chem. 2011, 22, 976-986, as well as dendritic polymer drug compositions, such as those described by Yin in U.S. Pat. No. 5,919,442.
Special protective chemistries were used during the termination step (Hsiue, Bioconj. Chem. 2006, 17, 781-786, U.S. Pat. No. 7,943,141, US Pub. No. 2011/0123453 and Zhang, et al., Macromol. 2009, 42(6)2215-2221). However, none of those approaches utilized an unprotected, multifunctional small terminating molecule for the in situ functionalization of linear POX polymers, which can significantly reduce production costs.
Assays and Microarrays
Since completion of the human genome project, it has become evident that elucidation of biological pathways and mechanisms at the protein level can be as important as studies at the genetic level because the former is more closely associated with disease and disease states, as well as the treatment thereof. With that strong demand, a new forum called proteomics developed and that art is a major focus of industrial and academic pursuits.
Currently, three major research areas of proteomics studies include drug discovery, high throughput screening and validation of new protein targets and drug leads. Tools include two dimensional (2-D) gel electrophoresis, mass spectrometry, and more recently, protein microarrays. In contrast to the lengthy 2-D gel procedures and tedious sample preparation (primarily separations) involved in mass spectrometry analysis, protein microarrays provide a quick, generally simple and low cost method to screen large amounts of proteins and the functions thereof. Therefore, microarrays are developing as desirable tools in proteomics.
However, protein-based microarray technology is far less developed than is gene microarray technology. The construction of a protein/antibody chip presents daunting challenges not encountered in the development of classical immunoassays or of DNA chips. For example, proteins are more sensitive to the environment than are nucleic acids. The hydrophobicity of many membrane, glass and plastic surfaces can cause protein denaturation destroying the structure and/or function thereof thereby rendering a protein reagent structurally and/or functionally inactive, which can result in lower assay sensitivity and higher signal-to-noise ratios. In other words, to construct a protein microarray, at least three issues must be addressed, protein denaturation, protein immobilization and protein orientation.
For example, a protein molecule often folds into a three-dimensional (3-D) structure in solution for and to maintain biological activity. On interaction with different solid surfaces, for example, during immobilization of proteins onto membranes, glass slides or micro/nanoparticles, the 3-D structure of the protein molecule often collapses thereby often destroying biological activity or at least functional structures. In addition, proteins often do not have the ability to adhere onto different surfaces.
To immobilize a protein on a surface, direct covalent linking reactions or electrostatic interactions (physical adsorption) often are employed. But, heterogeneous chemical reactions often are incomplete yielding undesired side products (i.e. incomplete modification of surfaces) and in some cases, also partially denatured proteins during different reaction stages.
Electrostatic interaction relies heavily on the isoelectric points of the proteins, as well as the pH of the buffer solutions.
Both approaches tend to yield irreproducible results due to the complexity of those procedures. Lot-to-lot reproducibility is, therefore, very poor.
As a result, there is interest in modifying solid substrates, but not the protein molecule, to obtain surfaces carrying biologically active protein. A variety of polymers, including PEI polymers, have been utilized as coating materials to alter the characteristics of solid surfaces for the construction of protein arrays, Wagner et al., U.S. Pat. No. 6,406,921.